The goal of Project I is to elucidate the mechanisms that link depletion of airway surface liquid volume to mucus accumulation and chronic infection. This Project proposes to utilize a human well-differentiated cell culture system to explore a series of hypotheses, and Specific Aim 1 is dedicated to the thorough characterization of secreted mucins in this system and their organization into a mucus layer, with comparison to a mucus sample obtained in vivo. After this characterization, the Project focuses its aims on testing a series of hypotheses with respect to the generation of CF lung disease. Specific Aim 2 will investigate the nature of the early interactions between the mucus layer and the cell surface glycocalyx that occur as a result of periciliary liquid depletion. This Aim will then go on to explore the hypothesis that no feedback exists between the absence of mucus transport (due to adhesive interactions of the mucus layer and cell surface glycocalyx), and goblet cell mucin secretion, so that mucus plaques and plugs form on CF airway surfaces. The Aim will also explore the corollaries that plaques and plugs wilt be more concentrated (thickened) on CF airways due to the absence of compensatory CI- secretion. Specific Aim 3 will explore the hypotheses that thickened mucus layers on CF airway surfaces provide a substrate for unique interactions with bacteria to promote the formation of biofilms, and conversely, provide a barrier to secondary lung defense mechanisms that typically are activated in response to infection, i.e., macrophage- and neutrophil-mediated bacterial killing. Finally, this Aim will explore mechanisms to restore volume to thickened mucus plaques and restore surface mucus transport, focusing on the hypothesis that irreversible phase transitions may require the addition of agents that attack mucin structure, in addition to volume restoration, to achieve this goal. Thus, the overall aims of the Project are designed to explore the behavior of mucins secreted into a water-depleted CF airway environment with respect to the steps of mucus stasis, mucus accumulation, persistent bacterial infection, and ultimately, methods to treat CF lung disease.